谷胱甘肽S-转移酶-KGDX融合蛋白的抗血小板作用  

The Antiplatelet Action of Glutathione S-transferase-KGDX Fusion Protein

作　　者：查艳萍[1] 秦永文[1] 荆清[1] 穆瑞斌[1] 

机构地区：[1]第二军医大学长海医院心内科,上海200433

出　　处：《现代临床医学生物工程学杂志》2001年第4期244-247,252,共5页Journal of Modern Clinical Medical Bioengineering

摘　　要：目的　观察重组GST -KGDX(谷胱甘肽S -转移酶 -赖 -甘 -天冬 -X)融合蛋白对血小板聚集的抑制作用 .方法　设计合成了两条编码KGDX(赖 -甘 -天冬 -X)的寡核苷酸链 ,长度为 36个碱基对 ,两端分别为BamHI酶和XcoI酶切位点 .将该基因插入原核高效表达载体PGEX - 4T - 1的tac启动子下游 ,获得重组质粒PGEX - 4T -1KGDX ,转化大肠杆菌DH5a,37℃诱导使重组子表达 .采用谷胱甘肽 -琼脂糖悬珠亲和层析法获得纯化蛋白 .用血小板凝聚仪检测活性 .结果　GST -KGDX融合蛋白具有可溶性 ,产量为 35mg/升培养基 ,表达量占菌体总蛋白 48.0 2 % ,容易从裂解菌中纯化得到 .体外实验 :GST、GST -KGDX融合蛋白均能抑制ADP诱导的人血小板聚集 ,GST -KGDX强于GST(p<0 .0 5或 <0 .0 1) ,其IC50 值分别为 4μmol/L、6 μmol/L .结论　 (1)GST、GST -KGDX融合蛋白在体外均能抑制ADP诱导的人血小板聚集 .(2 )大肠杆菌中能够表达出较高产量的GST -KGDX融合蛋白 ,为抗血小板因子提供了一个很好的蛋白质来源 .Objective To investigate the effect of GST-KGDX(glutathione S-transferase- Lys-Gly-Asp-X )frusion protein on platelet function in vitro.Methods The KGDX gene was assembled from 2 synthetic oligonucleotides,36 base paris in length,using BamHI and XhcoI restriction enzyme sites at the end of the gene for cloning into the expression vector PGEX4T-1.Expression of fusion protein was directed by the tac promoter.The Echerichia coli DH5a that contained the plasmid PGEX-4T-1-KGDX was induced by 37℃ heat induction.The fusion protein with (GST-KGDX) was purified in one step from the baterial lysate by glutathione-agarose beads for affinity chromatography.Inhibition of platelet Aggregation in platelet-rich plasma was determined for each of the peptides by a platelet aggreometer.Results Both GST and GST-KGDX were found to be soluble,abundant.Yields of 35mg/litre of cultures were obtained.The fused GST-KGDX protien was expressed in E.coli to a level of 48.02% of total celluar protein.GST-KGDX fusion protein inhibits ADP-induced human platelet aggregation more stronglier than GST(p<0.05 or <0.01),GST-KGDX has IC 50 of 4μmol/L,GST has IC 50 of 6μmol/L for human platelet.Conclusions (1)GST ?GST-KGDX fusion protein is functional in inhibition of platelet aggregation in vitro.(2)The bacteriactly produced fusion protein GST-KGDX can be properly used as an antithrombolitic agent.

关 键 词：基因重组 蛇毒 大肠杆菌 KPⅡb/Ⅲa拮抗剂 GST-KGDX融合蛋白 抗血小板作用 

分 类 号：R346[医药卫生—基础医学]